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European Journal of Human Genetics (2005) 13, 807–814& 2005 Nature Publishing Group All rights reserved 1018-4813/05 $30.00
An association study of the N-methyl-D-aspartatereceptor NR1 subunit gene (GRIN1) and NR2Bsubunit gene (GRIN2B) in schizophrenia withuniversal DNA microarray
Shengying Qin1,2,6, Xu Zhao1,2,6, Yuxi Pan1,2, Jianhua Liu3, Guoyin Feng4, Jingchun Fu5,Jiying Bao5, Zhizhou Zhang1,2 and Lin He*,1,2
1Bio-X Life Science Research Center, Shanghai Jiao Tong University, Shanghai 230030, PR China; 2Institute forNutritional Sciences, SIBS, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, PR China; 3School ofLife Science & Biotechnology, Shanghai Jiao Tong University, Shanghai, PR China; 4Shanghai Mental Health Center,Shanghai, PR China; 5Inner Mongolia Mental Health Center, Huhehaote, PR China
Dysfunction of the N-methyl-D-aspartate (NMDA) receptors has been implicated in the etiology ofschizophrenia based on psychotomimetic properties of several antagonists and on observation of geneticanimal models. To conduct association analysis of the NMDA receptors in the Chinese population, weexamined 16 reported SNPs across the NMDA receptor NR1 subunit gene (GRIN1) and NR2B subunit gene(GRIN2B), five of which were identified in the Chinese population. In this study, we combined universalDNA microarray and ligase detection reaction (LDR) for the purposes of association analysis, an approachwe considered to be highly specific as well as offering a potentially high throughput of SNP genotyping.The association study was performed using 253 Chinese patients with schizophrenia and 140 Chinesecontrol subjects. No significant frequency differences were found in the analysis of the alleles but somewere found in the haplotypes of the GRIN2B gene. The interactions between the GRIN1 and GRIN2B geneswere evaluated using the multifactor-dimensionality reduction (MDR) method, which showed a significantgenetic interaction between the G1001C in the GRIN1 gene and the T4197C and T5988C polymorphisms inthe GRIN2B gene. These findings suggest that the combined effects of the polymorphisms in the GRIN1 andGRIN2B genes might be involved in the etiology of schizophrenia.European Journal of Human Genetics (2005) 13, 807 – 814. doi:10.1038/sj.ejhg.5201418Published online 20 April 2005
Keywords: schizophrenia; universal DNA microarray; multifactor-dimensionality reduction (MDR)
transmission, plasticity and excitoxicity. Functional NMDA
As one type of the glutamate receptors in the glutamatergic
receptors are mainly composed of an NMDA receptor NR1
excitatory neurotransmitter system, N-methyl-D-aspartate
subunit (GRIN1) and one of the four NMDA receptor NR2
(NMDA) receptors play crucial roles in excitatory synaptic
subunits (GRIN2A, GRIN2B, GRIN2C and GRIN2D).1 – 3 Forseveral years, there has been considerable interest in thepossible role of the NMDA receptors in schizophrenia
*Correspondence: Dr L He, Bio-X Life Science Research Center, ShanghaiJiao Tong University, Hao Ran Building, PO Box 501, Shanghai 200030, PR
following the discovery that several noncompetitive
China. Tel: þ 86 21 64717740; Fax: þ 86 21 62822491;
antagonists of the NMDA receptors, such as phencyclidine
(PCP), ketamine and MK-801, were found to cause
6These authors contributed equally to this work.
psychotic conditions mimicking schizophrenia in humans
Received 30 August 2004; revised 20 January 2005; accepted 3 March2005
and laboratory animal models.4 – 8 The involvement of the
Association study in schizophrenia with microarray
NMDA receptors is also supported by genetic animal
SD ¼ 12.05) served as controls. The participants recruited in
models: for instance, knockdown mice expressing only
this study all provided written informed consent.
5% of normal levels of the essential GRIN1 subunit surviveto adulthood but display behavioral abnormalities, such as
elevated motor activity and stereotypy and deficiencies in
social and sexual interactions, which can be ameliorated by
We examined 11 SNPs (C112T, C113T, G716A, A750G,
treatment with haloperidol or clozapine.9 – 15
G1001C, A290G, G301A, G308A, A1970G, G2108A and
Mutation screening and association analysis for the
G6435A) in the GRIN1 gene and five SNPs (G366C,
NMDA receptors have been performed by several groups
C2664T, C3538T, T4197C and T5988C) in the GRIN2B
focusing mainly on the GRIN1 and GRIN2B genes.16 – 23 The
gene, which had been identified in previous studies.16,18,20
association of the variants in GRIN1 and GRIN2B with
The primer sets were designed to amplify the SNP-contain-
schizophrenia has been established in several reports,18,19
ing regions. The NCBI number of the SNP sites, corre-
but not replicated in others.16,22 Until now, GRIN1 and
sponding primer sequences and the sizes of the amplicons
GRIN2B have always been studied individually. However,
are presented in Supplementary material 1. The PCRs were
the functional NMDA receptors are heterodimer complexes
carried out on the PTC-100 DNA engine (MJ Research, USA)
that involve gene – gene interactions arising from interac-
in a total volume of 15 ml containing 10 ng of genomic
tions between receptor subunits and probably have a
DNA, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 – 3.0 mM
synergistic effect on the pathogenesis of schizophrenia.
MgCl2, 200 mM dNTP, 1 mM of each primer and 0.25 U Taq
There has been accumulating evidence supporting the
DNA polymerase. The cycling protocol consisted of
hypothesis that gene to gene interactions play an im-
denaturation at 951C for 1 min, followed by 30 – 35 cycles
portant role in susceptibility to common human dis-
at 951C for 30 s, 50 – 651C for 1 min, 721C for 1 min, and a
eases.24 In the light of this evidence, the present study
final extension at 721C for 10 min. Subsequently, the PCR
has focused on the GRIN1 and GRIN2B genes as a pilot
products were sequenced for SNP examination using ABI
PRISM 3100 DNA Sequencer (Applied Biosystems, USA).
DNA microarray technology has been advocated as one
For each SNP, 40 subjects were examined in both sense and
of the most powerful approaches for highly parallel, large-
scale SNP analysis. Several approaches for SNP genotypingwith DNA microarray have been designed.25 – 29 Recently,
the universal DNA microarray method mediated by ligase
In the universal microarray approach, five probes including
detection reaction (LDR) has been demonstrated to be a
two discriminating probes, one common probe and two
highly specific and sensitive assay for SNP detection.29 – 32
corresponding zip-codes, which are coupled to the slides at
We combined this approach with the slide self-activating
known locations, were designed for each SNP as described
method28,33 to establish a high-discrimination and cost-
in a previous study.29 In addition, the positive and negative
effective technology with significant promise for future
marker probes were designed for result evaluation. The
designed probes are summarized in Supplementary materi-
In the present study, we first examined the SNPs
al 1. The discriminating probe contains a zip-code
identified in previous studies across the GRIN1 and GRIN2B
complement sequence on the 50-end and the discriminat-
genes in the Chinese population, and then performed SNP
ing base on the 30-end. The common probe is phosphory-
genotyping using the universal microarray method to
lated on the 50-end and contains a fluorescent label (Cy3)
evaluate the single locus and haplotype associations. The
on the 30-end. The two probes are hybridized adjacent to
gene – gene interactions were examined with the multi-
one another on the template DNA and the nick between
factor-dimensionality reduction method (MDR), which can
the two probes can be sealed by the ligase only if there is
identify evidence for high-order gene to gene interactions
perfect complementarity at the junction. The ligated
in the absence of any statistically significant independent
products are hybridized to the zip-codes array. Only the
main effects with relatively small samples.34 – 36
spots corresponding to the SNP type are seen to givestronger fluorescent signals (Figure 1).
The standard microscope glass slides were first pre-
cleaned with 1 M NaOH overnight and washed several
times with distilled water. After a neutralization step in 1%
HCl and an extensive washing step in distilled water, the
All subjects were Han Chinese in origin. The 253 patients
pretreated glass slides were immersed in 2% 3-aminopro-
with schizophrenia (mean age of onset 25.45 years, mean
pyl-trimethoxysilane (Sigma, USA) for 15 min followed by
age at present 38.43 years, SD ¼ 10.9) were diagnosed
five washing steps in acetone. Subsequently, slides were
according to DSM-IV by two independent clinicians. The
baked at 1101C for 45 min, and then the slide surface was
140 unrelated healthy Han Chinese (mean age 43.37 years,
activated with 1,4-phenylene diisothiocyanate (Sigma,
Association study in schizophrenia with microarrayS Qin et al
Strategy for the universal microarray mediated by ligase detection reaction. The discriminating probe containing zip-code complement
sequence (czipa or czipb) and the common probe containing a fluorescent label can be ligated only if there is perfect complementarity at the junction.When hybridized to the universal array containing the zip-codes (zipa and zipb), the correspondent spots can be seen to give a stronger signal.
USA) according to Guo et al33 and Beier and Hoheisel.37
librium was measured between each of two locus and D0
The 50-terminal amino-modified zip-codes were dissolved
was the normalized linkage disequilibrim statistics.38
in a 400 mM sodium carbonate buffer (pH 9.0) to a final
Differences in allelic and separate haplotypic distributions
concentration of 20 mM and spotted using a GMS 417
were estimated by w2 test using EpiInfo 6.0 and the phasing
Arrayer (Genetic MicroSystems, USA) under a designed
of case and control haplotype was performed separately.
program. The slides were deactivated in 10% ammonium
The limit of significance was set to 0.05. The odds ratios
hydroxide solution. Finally, they were rinsed five times in
(OR) and their 95% confidence intervals were estimated by
distilled water and centrifuged for 1 min at 1000 r.p.m.
EpiInfo 6.0 for the effects of alleles. Difference in overall
The target DNA sequences were amplified using a
haplotype distribution was assessed by w2 test using
multiplex PCR method. After the completion of the
CLUMP1.639 with 300 000 simulations.
amplification, 1 ml of Proteinase K (20 mg/ml) was added,
The gene – gene interactions were analyzed by MDR
then heated at 701C for 10 min and quenched at 941C for
software. There are six general steps involved in imple-
15 min. The ligation reaction for each subject was carried
menting the MDR method for case – control study. The first
out in a final volume of 20 ml containing 20 mM Tris-HCl
step of MDR involves partitioning the data into some
(pH 7.6), 25 mM potassium acetate, 10 mM magnesium
number of equal parts for cross-validation. With 10-fold
acetate, 10 mM DTT, 1 mM NAD, 0.1% Triton X-100, 10 ml of
cross-validation in our study, the data are randomly
Multi-PCR product, 1 pmol of each discriminating oligo,
divided into 10 equal parts, and the MDR model is
1 pmol of each common probe and 0.5 ml of 40 U/ml Taq
developed on 9/10 of the data (training set) and then
DNA ligase (New England Biolabs, USA). The LDR was
tested on 1/10 of the remaining data (testing set). In step 2,
performed with 40 cycles of 941C for 30 s and 631C for
a set of n genetic factors is selected from the pool of all
factors. In step 3, the n factors and their multifactor classes
Following the LDR reaction, the solution was diluted to
or cells are represented in n-dimensional space. For
obtain 65 ml of hybridization mixture containing 5 Â SSC
example, for two polymorphisms, each with three geno-
and 0.1 mg/ml salmon sperm DNA. The mixture was then
types, there are nine two-locus genotype combinations.
quenched at 941C for 2 min and chilled on ice immedi-
Then, the ratio of the number of cases to the number of
ately. The area of the slide containing the oligo array was
controls is evaluated within each multifactor cell. In step 4,
sealed with Gene Frame slide chambers (ABgene, UK) and
each multifactor cell in n-dimensional space is labelled as
the mixture was then introduced. Hybridization was
high risk if the ratio of cases to controls meets or exceeds
carried out at 651C for 3 h. After removal of the chamber,
the threshold (T) of 1.0 and low risk if the threshold is not
the slide was washed for 2 min in 1 Â SSC, 0.1% SDS and
exceeded. In one recent report, it has been observed in
then for 1 min in 0.05 Â SSC by gently shaking at room
simulation studies that a threshold of 1.0 is optimal at this
temperature. Finally, the slide was spun at 1000 r.p.m. for
stage of MDR modelling for unbalanced data sets.40 In this
4 min and imaged on a GMS418 scanner (Genetic Micro-
way, a model for cases and controls is formed by pooling
Systems, USA). 10 mm resolution of 16-bit TIFF images was
those cells labelled high risk into one group and those cells
analyzed using the Genepix 2.0 software (Axon Instru-
labelled low risk into another group. This reduces the n-
ments, USA). The average signals after subtraction of local
dimensional model to one dimension. In step 5, all
background were used for calculating the signal ratios of
possible combinations of n factors are evaluated sequen-
spot pairs corresponding to different alleles.
tially for their ability to classify affected and unaffectedindividuals in the training set and the best n-factor model
is selected. In step 6, the independent test set from the
Haplotypes were generated using the program Arlequin
cross-validation is used to estimate the prediction error of
(http://anthropologie.unige.ch/arlequin). Haplotype fre-
the best model selected in step five. The prediction error is
quencies, linkage disequilibrium and Hardy – Weinberg
the proportion of subjects for whom an incorrect predic-
equilibrium were performed by Arlequin. Linkage disequi-
tion was made. Steps 1 to 6 are repeated 10 times with the
Association study in schizophrenia with microarray
data split into 10 different training and testing sets. The
that were difficult to sequence could be genotyped easily
number of times a particular set of factors is identified in
by the microarray procedure. The DNA targets quality
each possible 9/10 of the subjects is defined as cross-
required in this microarray procedure was not so strict as in
the sequencing method. As shown in Figure 2, the same
Once MDR identifies the best combination of factors, the
quality of the DNA target, which was genotyped only
final step is to determine which multifactor levels (eg
approximately by sequencing, could be discriminated
genotypes) are high risk and which are low risk using the
clearly by the microarray procedure.
entire data set. This final evaluation is conducted with a
In the present study, 16 reported polymorphisms (11 in
threshold ratio that is determined by the ratio of the
the GRIN1 gene and 5 in the GRIN2B gene) were selected
number of affected individuals in the dataset divided by
for examination in the Chinese population containing 20
the number of unaffected individuals in the dataset. Theprevious user-defined threshold ratio (T) is not used in thisfinal evaluation. Also, the MDR program can display thenumber of cases and controls for each multilocus –genotype combination. To make it clear, we showed thisas the percentage of it in the total subjects. Since the MDRprogram randomly shuffles the order of the observations inthe dataset using a random seed supplied by the user beforedividing the data into a training set and a testing set, weran the analysis 10 times using 10 different randomnumber seeds and the results were averaged in order toavoid spurious results due to chance divisions of the data.
This MDR procedure can be carried out for each possible
model size. In this study, we considered two-locus interac-tions through four-locus interactions. Single best modelsare selected from among each of the two-factor, three-factor and four-factor combinations. Among this set of bestmultifactor models, the combination of factors thatminimizes the prediction error and maximizes the cross-validation consistency is selected as the final best one.Finally, the statistical significance of the result wasdetermined by comparing the average prediction errorfrom the observed data with the distribution of averageprediction errors under the null hypothesis of no associa-tions derived empirically from 1000 permutations. Ourpermutation testing strategy was to randomize the case andcontrol labels in the original dataset 1000 times to create aset of permuted datasets. MDR can then be run on eachpermuted data set and the maximum cross-validationconsistency and minimum prediction error identified foreach were saved and used to create an empirical distribu-tion for estimation of a P-value The null hypothesis wasrejected when the upper-tail Monte Carlo P-value derivedfrom the permutation test was p0.05.
Characteristics evaluation of the DNA microarray
procedure. Each column of the array corresponds to a polymorphism
After the genotyping procedure, it was possible to validate
allele and contains five-spot replicates. Deposition scheme: 1 – 1001G;2 – 1001C; 3 – 366G; 4 – 366C; 5 – 2664C; 6 – 2664T; 7 – 4197T; 8 –
differences in sensitivity and specificity by direct sequen-
4197C; 9 – 5988T; 10 – 5988C; 11 – negative marker; 12 – positive
cing. We randomly selected 40 samples to be genotyped
marker. (a), (b) and (c) are the scanning results that, respectively,
using both methods. The result showed that the two
indicate that the five polymorphisms are all heterozygous, homo-
methods were completely consistent. It is clear that this
geneous and miscellaneous. (d) and (e) are the genotyping resultcomparisons between the microarrray and the sequencing method
microarray procedure demonstrates high specificity in
with the same quality template. The marked part of the picture is the
relation to genotype (Figure 2). Moreover, some regions
genotyping result of the C2664T locus of the same sample.
Association study in schizophrenia with microarrayS Qin et al
Allele distribution of the five polymorphisms in the GRIN1 and GRIN2B genes
aThe number of schizophrenics is 253.
patients and 20 controls. One variant in the GRIN1 gene
Estimation of linkage disequilibrium of the four
(G1001C) and four variants in the GRIN2B gene (G366C,
C2664T, T4197C and T5988C) were observed. Three of
them (G366C, C2664T and T4197C) were in coding
sequence, but were synonymous mutations. The fiveidentified SNPs were genotyped in 253 Chinese patients
with schizophrenia and 140 Chinese control subjects. The
distribution of these five SNPs was in Hardy – Weinberg
equilibrium (data not shown). The data for allele frequen-
cies are shown in Table 1. No significant differences in the
For each pair of SNPs, the standardized D0 is shown. D0 values X0.7
allele frequencies were observed between the patients andcontrols. Linkage disequilibrium between each pair ofGRIN2B SNPs is shown in Table 2. Three of the alleles instrong LD (C2664T, T4197C and T5988C) were used to
the null hypothesis of no association, it is highly unlikely
generate haplotypes. The haplotype frequencies of the
that a prediction error X30.772% will be observed for the
GRIN2B gene are shown in Table 3. We found that the
two-locus model and a prediction error X30.754% will be
frequency of the CCT haplotype (the C allele of the
observed for the three-locus model. As the MDR procedure
C2664T, the C allele of the T4197C, and the T allele of the
prefers the best model that has the minimum prediction
T5988C) was higher in controls (0.089) than in patients
error, the three-locus model, which included the G1001C
(0.022) (P ¼ 0.000146 after correction by the number of
polymorphism in the GRIN1 gene and the T4197C and
tests performed, w2 ¼ 18.79, OR ¼ 0.23). The difference of
T5899C polymorphisms in the GRIN2B gene, was regarded
CCC was nominally significant estimated by the w2 test
as the best model. Table 5 summarizes the three-locus –
(P ¼ 0.02, w2 ¼ 5.40, OR ¼ 1.54) using EpiInfo 6.0 and was
genotype combinations associated with high risk and with
not significant after the correction by the number of tests
low risk, along with the corresponding distribution of cases
performed (P ¼ 0.2). The overall haplotype distribution was
and of controls, for each multilocus – genotype combina-
(P ¼ 0.000031, w2 ¼ 23.60). With the MDR method, thegene – gene interactions were examined, and the results foreach number of factors considered are summarized inTable 4. All the models had the same cross-validation
consistency of 10.0 and the permutation testing showed
The microarray procedure used in this study was demon-
that the prediction errors of the two-locus and three-locus
strated to be reliable by comparing with sequencing
models are both significant at the 0.001 level. Thus, under
method in the examination of 40 subjects. The universal
Association study in schizophrenia with microarray
Frequencies of estimated haplotypes composed of the three polymorphisms of the GRIN2B gene
aThe haplotype frequencies defined by C2664T, T4197C and T5988C were determined. Only haplotypes with a frequency greater than 2% are listed.
bValues in parentheses are P-values corrected by the number of tests performed. P ¼ 0.000031 (w2 ¼ 23.60) for difference in overall haplotypedistribution.
Results of the MDR analysis of the dataset
SNPs included in the best candidate model
*Po0.001 based on 1000 permutations.
Distribution of high-risk and low-risk genotypes in the best three-locus model
microarray platform can be used for any group of SNPs and
selection acting against molecular diversity during human
only the LDR primers require to be designed. Compared
evolution. Of all the identified variants in both GRIN1 and
with other gene-specific arrays, the microarray procedure
GRIN2B genes, three are located in protein coding
gives researchers freedom to detect specified polymorphic
sequences, but only as synonymous mutations. Moreover,
loci based on the same universal array and avoids the need
one polymorphism G1001C seemed to have functional
to optimize the operation condition each time. Further-
significance. It is located in the promoter region of GRIN1
more, this approach can readily distinguish the poly-
and could alter a consensus sequence for the p50 subunit of
morphism types of insertions and deletions in repeat
the transcription factor NF-kB.19 The association analysis
of G1001C polymorphism in the GRIN1 gene has been
On examination of the SNPs, only one of the 11 reported
done by several groups,19,21 – 23 but only Begni et al19
SNPs existed in the GRIN1 gene in our sample, which is
detected a significant result in the Italian population. In
perhaps due to ethnic differences. The lower rate of
the previous association study for the four polymorphisms
nonsynonymous SNPs in this gene, which has also been
(G366C, C2664T, T4197C and T5988C) in the GRIN2B
reported by other groups,16,17 may be mainly due to
gene, Ohtsuki et al18 observed a significant difference in
Association study in schizophrenia with microarrayS Qin et al
the allele frequency of the G366C polymorphism between
interaction between the G1001C in the GRIN1 gene and
patients and control individuals in the Japanese popula-
the T4197C and T5988C polymorphisms in the GRIN2B
tion. Also, their haplotype analysis showed a borderline
gene. To our knowledge, this is the first report of the
significant result for the overall haplotype distribution.18
interaction between the two subunit genes of the NMDA
However, the significant result of G366C was not replicated
receptors associated with schizophrenia.
in the analysis on a sample of Caucasian patients.16 In our
In conclusion, using the universal microarray procedure
study, no significant frequency differences of the five
mediated by LDR combined with effective MDR statistical
alleles (G1001C, G366C, C2664T, T4197C, and T5988C)
analysis, our study provided the possibility that the
between schizophrenic patients and controls were ob-
combinational effects of the polymorphisms in the GRIN1
served. However, in the low-frequency haplotype (CCT)
and GRIN2B genes are associated with schizophrenia in the
and the overall haplotype distribution of the GRIN2B gene,
Chinese population. However, as case – control studies are
we did find evidence of association with schizophrenia.
susceptible to positive and negative artefacts from un-
Moreover, all the three SNPs contained in the haplotype of
known population stratification, it is necessary to validate
our study are also included in the significant haplotype of
or replicate our association results in other ethnic groups.
the previous study by Ohtsuki et al, which furthersupported possible association of the haplotype in GRIN2Bgene with schizophrenia. Since this association has been
found in a relatively small sample size (253 cases and 140
This work was supported by grants from the national 973 and 863
controls) involving a low-frequency haplotype, it is
programs, the National Natural Science Foundation of China, the
susceptible to a false-positive result and further study with
Shanghai Municipal Commission for Science and Technology, and theKey Project of the Ministry of Education (No. 03066). We are grateful
to the Office of Technology Transfer & Enterprise Development of the
There is a growing awareness that the complex disorders
Vanderbilt University for providing the MDR software and to Dr Lance
may be genetically attributable to complex interactions
among multiple genes. Many studies have observed thiscombination effect in a number of complex disorders.34,40 –44 The two subunits of the NMDA receptors have different
functions. The GRIN1 subunit possesses the characteristic
1 Hollmann M, Heinemann S: Cloned glutamate receptors. Annu
ion channel properties and the specificity of the NMDA
2 Nakanishi S, Masu M: Molecular diversity and functions of
receptors arises from the GRIN2 subunits, which display
glutamate receptors. Annu Rev Biophys Biomol Struct 1994; 23:
different regional and temporal expression patterns.1 – 3 We
hypothesized that gene – gene interactions across the two
3 Moriyoshi K, Masu M, Ishii T, Shigemoto R, Mizuno N, Nakanishi
S: Molecular cloning and characterization of the rat NMDA
subunit genes probably contributes to the pathogenesis of
receptor. Nature 1991; 354: 31 – 37.
schizophrenia although the specific SNPs alone do not
4 Luby ED, Cohen BD, Rosenbaum G, Gottlieb JS, Kelley R: Study of
show any association with the disorder. Using the MDR
a new schizophrenomimetic drug; sernyl. AMA Arch Neurol
method, we were able to detect interactions in the absence
5 Javitt DC, Zukin SR: Recent advances in the phencyclidine model
of main effects, where logistic regression and other
of schizophrenia. Am J Psychiatry 1991; 148: 1301 – 1308.
approaches may not be able to do so.34 Application of
6 Ellison G: The N-methyl-D-aspartate antagonists phencyclidine,
MDR to case – control data sets has routinely yielded
ketamine and dizocilpine as both behavioral and anatomicalmodels of the dementias. Brain Res Brain Res Rev 1995; 20: 250 –
statistically significant interactions in various common
human diseases, such as sporadic breast cancer, essential
7 Malhotra AK, Pinals DA, Adler CM et al: Ketamine-induced
hypertension, atrial fibrillation and type II diabetes
exacerbation of psychotic symptoms and cognitive impairment
mellitus.34,40,43,44 In our MDR analysis, although the
prediction errors of the two-locus and three-locus models
8 Abi-Saab WM, D’Souza DC, Moghaddam B, Krystal JH: The
had the same level of significance, the three-locus model
NMDA antagonist model for schizophrenia: promise and pitfalls.
had a lower prediction error (30.754%) than that in the
Pharmacopsychiatry 1998; 31 (Suppl 2): 104 – 109.
two-locus model. Furthermore, the three loci in the three-
9 Li Y, Erzurumlu RS, Chen C, Jhaveri S, Tonegawa S: Whisker-
related neuronal patterns fail to develop in the trigeminal
locus model were located within the two different subunit
brainstem nuclei of NMDAR1 knockout mice. Cell 1994; 76:
genes, which may display the gene to gene interactions
associated with schizophrenia and the two-locus model are
10 Forrest D, Yuzaki M, Soares HD et al: Targeted disruption of
NMDA receptor 1 gene abolishes NMDA response and results in
included in the three-locus model. Thus, we selected the
neonatal death. Neuron 1994; 13: 325 – 338.
three-locus model as the best model. However, the MDR
11 Ikeda K, Araki K, Takayama C et al: Reduced spontaneous activity
program could not statistically compare the two models to
of mice defective in the epsilon 4 subunit of the NMDA receptor
confirm that the three-locus model is more significantly
channel. Brain Res Mol Brain Res 1995; 33: 61 – 71.
12 Sakimura K, Kutsuwada T, Ito I et al: Reduced hippocampal LTP
associated than the two-locus model, which is a limitation
and spatial learning in mice lacking NMDA receptor epsilon 1
of the present study. The best model showed a genetic
subunit. Nature 1995; 373: 151 – 155.
Association study in schizophrenia with microarray
13 Ebralidze AK, Rossi DJ, Tonegawa S, Slater NT: Modification of
primer elongation reaction on oligonucleotide microarrays.
NMDA receptor channels and synaptic transmission by targeted
disruption of the NR2C gene. J Neurosci 1996; 16: 5014 – 5025.
29 Gerry NP, Witowski NE, Day J, Hammer RP, Barany G, Barany F:
14 Kutsuwada T, Sakimura K, Manabe T et al: Impairment of suckling
Universal DNA microarray method for multiplex detection of low
response, trigeminal neuronal pattern formation, and hippocam-
abundance point mutations. J Mol Biol 1999; 292: 251 – 262.
pal LTD in NMDA receptor epsilon 2 subunit mutant mice.
30 Consolandi C, Busti E, Pera C et al: Detection of HLA
polymorphisms by ligase detection reaction and a universal array
15 Das S, Sasaki YF, Rothe T et al: Increased NMDA current and spine
format: a pilot study for low resolution genotyping. Hum
density in mice lacking the NMDA receptor subunit NR3A. Nature
31 Favis R, Day JP, Gerry NP, Phelan C, Narod S, Barany F: Universal
16 Williams NM, Bowen T, Spurlock G et al: Determination of the
DNA array detection of small insertions and deletions in BRCA1
genomic structure and mutation screening in schizophrenic
and BRCA2. Nat Biotechnol 2000; 18: 561 – 564.
individuals for five subunits of the N-methyl-D-aspartate gluta-
32 Busti E, Bordoni R, Castiglioni B et al: Bacterial discrimination by
mate receptor. Mol Psychiatry 2002; 7: 508 – 514.
means of a universal array approach mediated by LDR (ligase
17 Sakurai K, Toru M, Yamakawa-Kobayashi K, Arinami T: Mutation
detection reaction). BMC Microbiol 2002; 2: 27.
analysis of the N-methyl-D-aspartate receptor NR1 subunit gene
33 Guo Z, Guilfoyle RA, Thiel AJ, Wang R, Smith LM: Direct
(GRIN1) in schizophrenia. Neurosci Lett 2000; 296: 168 – 170.
fluorescence analysis of genetic polymorphisms by hybridization
18 Ohtsuki T, Sakurai K, Dou H, Toru M, Yamakawa-Kobayashi K,
with oligonucleotide arrays on glass supports. Nucleic Acids Res
Arinami T: Mutation analysis of the NMDAR2B (GRIN2B) gene in
schizophrenia. Mol Psychiatry 2001; 6: 211 – 216.
34 Ritchie MD, Hahn LW, Roodi N et al: Multifactor-dimensionality
19 Begni S, Moraschi S, Bignotti S et al: Association between the
reduction reveals high-order interactions among estrogen-meta-
G1001C polymorphism in the GRIN1 gene promoter region and
bolism genes in sporadic breast cancer. Am J Hum Genet 2001; 69:
schizophrenia. Biol Psychiatry 2003; 53: 617 – 619.
20 Rice SR, Niu N, Berman DB, Heston LL, Sobell JL: Identification of
35 Hahn LW, Ritchie MD, Moore JH: Multifactor dimensionality
single nucleotide polymorphisms (SNPs) and other sequence
reduction software for detecting gene-gene and gene-environ-
changes and estimation of nucleotide diversity in coding and
ment interactions. Bioinformatics 2003; 19: 376 – 382.
flanking regions of the NMDAR1 receptor gene in schizophrenic
36 Ritchie MD, Hahn LW, Moore JH: Power of multifactor dimen-
patients. Mol Psychiatry 2001; 6: 274 – 284.
sionality reduction for detecting gene – gene interactions in the
21 Tani A, Kikuta R, Itoh K et al: Polymorphism analysis of the
presence of genotyping error, missing data, phenocopy, and
upstream region of the human N-methyl-D-aspartate receptor
genetic heterogeneity. Genet Epidemiol 2003; 24: 150 – 157.
subunit NR1 gene (GRIN1): implications for schizophrenia.
37 Beier M, Hoheisel JD: Versatile derivatisation of solid support
media for covalent bonding on DNA-microchips. Nucleic Acids Res
22 Martucci L, Wong AH, Trakalo J et al: N-methyl-D-aspartate
receptor NR1 subunit gene (GRIN1) in schizophrenia: TDT and
38 Jorde LB: Linkage disequilibrium and the search for complex
case-control analyses. Am J Med Genet 2003; 119B: 24 – 27.
disease genes. Genome Res 2000; 10: 1435 – 1444.
23 Hung CC, Chen HY, Chen CH: Systematic mutation analysis of
39 Sham PC, Curtis D: Monte Carlo tests for associations between
the human glutamate receptor, ionotropic, N-methyl-D-aspartate
disease and alleles at highly polymorphic loci. Ann Hum Genet
1 gene(GRIN1) in schizophrenic patients. Psychiatr Genet 2002;
40 Cho YM, Ritchie MD, Moore JH et al: Multifactor-dimensionality
24 Moore JH: The ubiquitous nature of epistasis in determining
reduction shows a two-locus interaction associated with Type 2
susceptibility to common human diseases. Hum Hered 2003; 56:
diabetes mellitus. Diabetologia 2004; 47: 549 – 554.
41 Xu Q, Jia YB, Zhang BY et al: Association study of an SNP
25 Pastinen T, Kurg A, Metspalu A, Peltonen L, Syvanen AC:
combination pattern in the dopaminergic pathway in paranoid
Minisequencing: a specific tool for DNA analysis and diagnostics
schizophrenia: a novel strategy for complex disorders. Mol
on oligonucleotide arrays. Genome Res 1997; 7: 606 – 614.
26 Southern EM, Maskos U, Elder JK: Analyzing and comparing
42 Carrasquillo MM, McCallion AS, Puffenberger EG, Kashuk CS,
nucleic acid sequences by hybridization to arrays of oligonucleo-
Nouri N, Chakravarti A: Genome-wide association study and
tides: evaluation using experimental models. Genomics 1992; 13:
mouse model identify interaction between RET and EDNRB
pathways in Hirschsprung disease. Nat Genet 2002; 32: 237 – 244.
27 Gunderson KL, Huang XC, Morris MS, Lipshutz RJ, Lockhart DJ,
43 Moore JH, Williams SM: New strategies for identifying gene – gene
Chee MS: Mutation detection by ligation to complete n-mer DNA
interactions in hypertension. Ann Med 2002; 34: 88 – 95.
arrays. Genome Res 1998; 8: 1142 – 1153.
44 Tsai CT, Lai LP, Lin JL et al: Renin – angiotensin system gene
28 Erdogan F, Kirchner R, Mann W, Ropers HH, Nuber UA: Detection
polymorphisms and atrial fibrillation. Circulation 2004; 109:
of mitochondrial single nucleotide polymorphisms using a
Supplementary Information accompanies the paper on European Journal of Human Genetics website (http://www.nature.com/ejhg)